The present invention relates to peptides of one or more portions of the human chorionic gonadotropin xcex2-chain as well as methods for treatment and prevention of diseases, including HIV infection, cancer and wasting syndrome, and methods of promoting hematopoiesis using human chorionic gonadotropin, employing the xcex2-chain of human chorionic gonadotropin, peptides containing a sequence of one or more portions of the xcex2-chain of human chorionic gonadotropin and derivatives and analogues thereof. The invention further relates to fractions of sources and or preparations of human chorionic gonadotropin, such as fractions of human early pregnancy urine, which fractions have anti-HIV activity, anti-cancer activity, anti-wasting activity, and/or pro-hematopoietic activity. The present invention further relates to pharmaceutical compositions for treating and/or preventing HIV infection, cancer, and/or wasting, and/or for promoting hematopoiesis.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F., et al., 1986, Science 233:343-346; Guyader, M., et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. In humans, HIV replication occurs prominently in CD4+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N., et al., 1984, RNA Tumor Viruses, Weiss, R., et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439).
The HIV viral particle comprises a viral core, composed in part of capsid proteins, together with the viral RNA genome and those enzymes required for early replicative events. Myristylated gag protein forms an outer shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 16.0 kilodalton precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M., and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD4+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish, A., et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (McDougal, J. S., et al., 1986, Science 231:382-385; Maddon, P. J., et al., 1986, Cell 47:333-348), explaining HIV""s tropism for CD4+ cells, while gp4l anchors the envelope glycoprotein complex in the viral membrane. While these virus:cell interactions are necessary for infection, there is evidence that additional virus:cell interactions are also required.
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective Therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H., et al., 1991, FASEB J. 5:2369-2381). Many viral targets for intervention with HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H., et al., 1991, Science 249:1533-1544).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddI), dideoxycytidine (ddC) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). For example, impressive results have recently been obtained with a combination of AZT, ddI, 3TC and ritonavir (Perelson, A. S., et al., 1996, Science 15:1582-1586). However, it is likely that long-term use of combinations of these chemicals will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8+ T cells, which are essential to the control of HIV, via killer cell activity (Blazevic, V., et al., 1995, AIDS Res. Hum. Retroviruses 11:1335-1342) and by the release of suppressive factors, notably the chemokines Rantes, MIP-1xcex1 and MIP-1xcex2 (Cocchi, F., et al., 1995, Science 270:1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses 10:S77-82). It is thought that such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4+ T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith, D. H., et al., 1987, Science 238:1704-707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar, E., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley, R., et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O., et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R., et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). Recently, chemokines produced by CD8+ T cells have been implicated in suppression of HIV infection (Paul, W. E., 1994, Cell 82:177; Bolognesi, D. P., 1993, Semin. Immunol. 5:203). The chemokines RANTES, MIP-1xcex1 and MIP-1xcex2, which are secreted by CD8+ T cells, were shown to suppress HIV-1 p24 antigen production in cells infected with HIV-1 or HIV-2 isolates in vitro (Cocchi, F, et al., 1995, Science 270:1811-1815). Thus, these and other chemokines may prove useful in therapies for HIV infection. The clinical outcome, however, of all these and other candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins appear to be the most promising candidates to act as antigens for anti-HIV vaccinen development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L., et al., U.S. Pat. No. 5,141,867; Saith, G., et al., WO92/22,654; Shafferman, A., WO91/09,872, Formoso, C., et al., WO90/07,119. Vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
The morphologically recognizable and functionally capable cells circulating in blood include erythrocytes, neutrophilic, eosinophilic, and basophilic granulocytes, B-, T-, non B-, non T-lymphocytes, and platelets. These mature hematopoietic cells derive from and are replaced, on demand, by morphologically recognizable dividing precursor cells for the respective lineages such as erythroblasts for the erythrocyte series, myeloblasts, promyelocytes and myelocytes for the granulocyte series, and megakaryocytes for the platelets. The precursor cells derive from more primitive cells that can simplistically be divided into two major subgroups: stem cells and progenitor cells (for review, see Broxmeyer, H. E., 1983, xe2x80x9cColony Assays of Hematopoietic Progenitor Cells and Correlations to Clinical Situations,xe2x80x9d CRC Critical Reviews in Oncology/Hematology 1:227-257).
The definitions of stem and progenitor cells are operational and depend on functional, rather than on morphological, criteria. Stem cells have extensive self-renewal or self-maintenance capacity (Lajtha, L. G., 1979, Differentiation 14:23), a necessity since absence or depletion of these cells could result in the complete depletion of one or more cell lineages, events that would lead within a short time to disease and death. Some of the stem cells differentiate upon need, but some stem cells or their daughter cells produce other stem cells to maintain the pool of these cells. Thus, in addition to maintaining their own kind, pluripotential stem cells are capable of differentiation into several sub-lines of progenitor cells with more limited self-renewal capacity or no self-renewal capacity. These progenitor cells ultimately give rise to the morphologically recognizable precursor cells. The progenitor cells are capable of proliferating and differentiating along one, or more than one, of the myeloid differentiation pathways (Lajtha, L. G. (Rapporteur), 1979, Blood Cells 5:447).
A variety of infectious agents, genetic abnormalities and environmental factors can cause a deficiency in one or more hematopoietic cell types. For example, hematological abnormalities have been observed in HIV-1 infected individuals (the human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503)), particularly in the late stages of disease (Lunardi-Iskandar, Y. et al., 1989, J. Clin. Invest 83:610-615). These abnormalities include a reduction in CD4+ T cells as well as cytopenias of one or more hematopoietic lineages, often associated with bone marrow morphologic abnormalities and deficient progenitor cell growth (Lunardi-Iskandar, Y. et al., 1989, J. Clin. Invest 83:610-615; Louache, F. et al., 1992, Blood 180:2991-2999). Idiopathic thrombocytopenic purpura (ITP), characterized by significant reduction in platelet numbers, often afflicts subjects infected with HIV (Ballem, P. J. et al., 1992, N. Engl. J. Med. 327:1779). The destruction of platelets in sufferers of ITP appears to be mediated by platelet associated autoantibodies (Berchtold, P. and Wenger, M., 1993, Blood 81:1246; Ballem, P. J. et al., 1987, J. Clin. Invest. 80:33). Thus, because management of ITP generally involves immunosuppression, treatment of ITP in HIV infected patients is complicated as administration of immunosuppressive drugs is extremely detrimental in HIV infection.
Additionally, chemotherapy and radiation therapy used in the treatment of cancer and certain immunological disorders can cause pancytopenias or combinations of anemia, neutropenia and thrombocytopenia. Thus, the increase or replacement of hematopoietic cells is often crucial to the success of such treatments. (For a general discussion of hematological disorders and their causes, see e.g., xe2x80x9cHematologyxe2x80x9d in Scientific American Medicine, E. Rubenstein and D. Federman, eds., volume 2, chapter 5, Scientific American, New York (1996)).
Furthermore, aplastic anemia presents a serious clinical condition as the overall mortality of all patients with aplastic anemias, in the absence of stem cell therapy, is high. Approximately 60-75% of individuals suffering from the disorder die within 12 months, in the absence of new stem cells. The overall incidence of these diseases is approximately 25 new cases per million persons per year. Although it is extremely unlikely that a single pathogenic mechanism accounts for all aplastic anemias, it is clear that provision of new hematopoietic stem cells is usually sufficient to allow permanent recovery, since transplantation of patients with aplastic anemia with bone marrow obtained from identical twins (i.e., syngeneic) (Pillow, R. P., et al., 1966, N. Engl. J. Med. 275:94-97) or from HLA-identical siblings (i.e., allogeneic) (Thomas, E. D., et al., Feb. 5, 1972, The Lancet, pp. 284-289) can fully correct the disease. However, some patients with aplastic anemia reject the transplanted marrow. This complication is particularly common among patients who have been immunologically sensitized as a result of multiple therapeutic blood transfusions.
The current therapy available for many hematological disorders as well as the destruction of the endogenous hematopoietic cells caused by chemotherapy or radiotherapy is bone marrow transplantation. However, use of bone marrow transplantation is severely restricted since it is extremely rare to have perfectly matched (genetically identical) donors, except in cases where an identical twin is available or where bone marrow cells of a patient in remission are stored in a viable frozen state. Except in such autologous cases, there is an inevitable genetic mismatch of some degree, which entails serious and sometimes lethal complications. These complications are two-fold. First the patient is usually immunologically incapacitated by drugs beforehand, in order to avoid immune rejection of the foreign bone marrow cells(host versus graft reaction). Second, when and if the donated bone marrow cells become established, they can attack the patient (graft versus host disease), who is recognized as foreign. Even with closely matched family donors, these complications of partial mismatching are the cause of substantial mortality and morbidity directly due to bone marrow transplantation from a genetically different individual.
Peripheral blood has also been investigated as a source of stem cells for hematopoietic reconstitution (Nothdurtt, W., et al., 1977, Scand. J. Haematol. 19:470-481; Sarpel, S. C., et al., 1979, Exp. Hematol. 7:113-120; Ragharachar, A., et al., 1983, J. Cell. Biochem. Suppl. 7A:78; Juttner, C. A., et al., 1985, Brit. J. Haematol. 61:739-745; Abrams, R. A., et al., 1983. J. Cell. Biochem. Suppl. 7A:53; Prummer, O., et al., 1985, Exp. Hematol. 13:891-898). In some studies, promising results have been obtained for patients with various leukemias (Reiffers, J., et al., 1986, Exp. Hematol. 14:312-315; Goldman, J. M., et al., 1980, Br. J. Haematol. 45:223-231; Tilly, H., et al., Jul. 19, 1986, The Lancet, pp. 154-155; see also To, L. B. and Juttner, C. A., 1987, Brit. J. Haematol. 66: 285-288, and references cited therein); and with lymphoma (Korbling, M., et al., 1986, Blood 67:529-532). Other studies using peripheral blood, however, have failed to effect reconstitution (Hershko, C., et al., 1979, The Lancet 1:945-947; Ochs, H. D., et al., 1981, Pediatr. Res. 15:601). Studies have also investigated the use of fetal liver cell transplantation (Cain, G. R., et al., 1986, Transplantation 41:32-25; Ochs, H. D., et al., 1981, Pediatr. Res. 15:601; Paige, C. J., et al., 1981, J. Exp. Med. 153:154-165; Touraine, J. L., 1980, Excerpta Med. 514:27:7; Touraine, J. L., 1983, Birth Defects 19:139; see also Good, R. A., et al., 1983, Cellular Immunol. 82:44-45 and references cited therein) or neonatal spleen cell transplantation (Yunis, E. J., et al; 1974, Proc. Natl. Acad Sci. U.S.A. 72:4100) as stem cell sources for hematopoietic reconstitution. Cells of neonatal thymus have also been transplanted in immune reconstitution experiments (Vickery, A. C., et al., 1983, J. Parasitol. 69(3):478-485; Hirokawa, K., et al., 1982, Clin. Immunol. Immunopathol. 22:297-304).
Clearly, there is a tremendous need for methods of expanding blood cells in vitro or therapies which increase the production of hematopoietic cells in vivo.
Wasting syndrome is a serious clinical problem characterized by a decrease in body mass of more than 10% from baseline body weight and a disproportionate loss of body mass with respect to body fat (Weinroth et al., 1995, Infectious Agents and Disease 4:76-94; Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275). Thus, wasting is distinguished from starvation in which higher levels of body fat than body cell mass are depleted (Kotler et al., 1985, Am J. Clin. Nutr. 42:1255-1265; Cahill, 1970, N. Engl. J. Med. 282:668-675). Wasting is associated with a variety of conditions, including HIV infection (human immunodeficiency virus (HIV) has been implicated in acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503)), other infectious diseases, sepsis, cancer, chronic cardiovascular disease and diarrhea (Kotler et al., 1989, Am. J. Clin. Nutr. 50:444-447; Heymsfield et al., 1982, Am. J. Clin. Nutr. 36:680-690). Importantly, wasting is a significant factor in the mortality of patients suffuering from infections or cancer. In fact, body cell mass depletion has a linear relationship to time of survival in AIDS patients (Kotler et al., 1989, Am. J. Clin. Nutr. 50:444-447).
The cause of wasting syndrome in AIDS and other conditions is unclear and is most likely multifactorial. Metabolic abnormalities, irregular levels of hormones and cytokines, and malabsorption have all been implicated in wasting syndrome. Not all AIDS patients suffer from wasting, suggesting that the cause of the wasting is not HIV itself. Most cases of HIV associated wasting syndrome are apparently caused by complications of AIDS, such as secondary infections and gastrointestinal disease (Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275).
Current and potential therapies for wasting syndromes include nutritional support, appetite enhancers such as dronabinol and megestrol acetate, anabolic therapies, such as growth hormone, and cytokine inhibitors. However, mixed results have been obtained with nutritional support and appetite enhancers in that patients tended to gain only fat and not overall body mass. Administration of growth hormone, and cytokine inhibitors are still being tested and may pose a risk of side effects (Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275; Weinroth et al., 1995, Infectious Agents and Disease 4:76-94).
Thus, treatment of wasting is critical to the survival and well-being of patients suffering from serious diseases such as cancer and AIDS; thus, there is a need for safe and effective therapies for wasting syndrome associated with cancer, AIDS and other infectious diseases.
A neoplasm, or tumor, is a neoplastic mass resulting from abnormal uncontrolled cell growth, which may cause swelling on the body surface, and which can be benign or malignant. Benign tumors generally remain localized. Malignant tumors, are collectively termed cancers. The term xe2x80x9cmalignantxe2x80x9d generally means that the tumor can invade and destroy neighboring body structures and spread to distant sites to cause death (for review, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Treatment options, such as surgery, chemotherapy and radiation treatment, are either ineffective or present serious side effects. Thus, there is a need for development of new drugs for the treatment of cancer.
Kaposi""s Sarcoma (KS) is a rare type of cancer, the incidence of which is greatly increased in HIV infected people (Lunardi-Iskandar, Y., et al., 1995, Nature 375:64-68; Friedman-Kien, A. E., et al., 1981. J. Am. Acad. Dermatol. 5:468-473). The tumors appear to be comprised of hyperplastic cells derived from vascular endothelial cells (Nakamura, S., et al., 1988, Science 242:426-430; Ensoli, B., et al, 1989, Science 243:223-226; Salahuddin, S. Z., et al., 1988, Science 242:430-433; Masood, R., et al., 1994, AIDS Res. Hum. Retroviruses 10:969-976; Lunardi-Iskandar, Y., et al., 1995, JNCI 88:450-454). In some cases, neoplastic cells with chromosomal abnormalities are also present in the tumors (Lunardi-Iskandar, Y., et al., 1995, JNCI 87:974-981; Delli-Bovi, P., et al., 1986, Cancer Res. 46:6333-6338; Siegal, B., et al., 1990, Cancer 65:492-498; Yunis, J. J., 1983, Science 221:227-236; Popescu, N. C., et al., 1995, JNCI 88:450-454). Therapies for KS include radiotherapy, xcex1-interferon and systemic chemotherapy (Chak, L. Y., et al., 1988, J. Clin. Oncol. 6:863-7; Evans, L. M., et al., 1991, J. Immunother. 10:39-50; Kovas, J., et al., 1990, Ann. Intern. Med. 112:812-21; Gelmann, E. D., et al., 1987, Am. J. Med. 82:456-62; Gill, P. S., et al., 1991, Am. J. Med. 90:427-33; Gill, P. S., et al., 1990; Am. J. Clin. Oncol. 13:315-9; Gill, P. S., et al., 1994, AIDS 8:1695-9). However, hematological and non-hematological toxicities limit the prolonged use of chemotherapy and xcex1-interferon in conjunction with anti-retroviral agents commonly used in the treatment of AIDS (Kovas, J., et al., 1990, Ann. Intern. Med. 112:812-21; Gill, P. S., et al., 1991, Am. J. Med. 90:427-33; Gill, P. S., et al., 1994, AIDS 8:1695-9). Thus, new drugs, preferably drugs compatible with AIDS therapeutics, are needed for the treatment of KS.
Human chorionic gonadotropin (hCG), which is required for the maintenance of pregnancy, is a member of the glycoprotein hormone family. The glycoprotein hormones, which also include follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH), consist of two sub-units, xcex1 and xcex2. These subunits are non-covalently linked to form a heterodimer, and heterodimer formation has been shown to be required for receptor binding. Within a particular species, the xcex1-subunits are identical among the glycoprotein hormones while the xcex2-subunits differ and determine the receptor binding specificity of the particular hormone (Kornyei, J. L., et al., 1993, Biol. Reprod. 49:1149). The xcex2-subunits of the glycoprotein hormones exhibit a high degree of sequence similarity within the N-terminal 114 amino acids. LH is the most similar to hCG with 85% sequence homology within the first 114 amino acids, and both proteins bind the same receptor. hCG, however, contains a C-terminal extension not present in the other glycoprotein xcex2-chains (Lapthorn, A. J., et al., 1994, Science 369:455-461).
From the three dimensional crystal structure of hCG, it has been determined that hCG, like the growth factors nerve growth factor (NGF), transforming growth factory-xcex2 (TGF-xcex2) and platelet-derived growth factor-xcex2 (PDGF-xcex2), is a cysteine-knot glycoprotein. Proteins containing such a cysteine-knot motif have at least three disulfide bridges, two of which join adjacent anti-parallel strands of the peptide, thus, forming a ring, and one of which joins the peptide chain through the ring. Particular structures in the hCG xcex2-chain include the determinant loop sequence (xcex293-100) which has been implicated in subunit association and the longest intercysfeine loop (xcex238-57) which may play a role6in receptor binding. Residues 47-53 appear to be exposed at the surface of this inter-cysteine loop (Lapthorn et al., 1994, Nature 369:455-461).
Previously, purified preparations of heterodimeric hCG have been shown to reduce the reverse transcriptase activity in HIV-1 infected lymphocytes and monocytes in culture (Bourinbaiar, A. S., and Nagorny, R., 1992, FEMS Microbiology Letters 96:27-30) and to prevent transmission of HIV from lymphocytes to trophoblasts in vitro (Bourinbaiar, A. S., and Nagorny, R., 1992, FEBS Letters 309:82-84). Additionally, the xcex2-subunit of hCG (xcex2-hCG) has been demonstrated to reduce HIV production in lymphocytes at doses from 100 pg/ml to 100 xcexcg/ml and in monocytes at doses up to approximately 10 xcexcg/ml, with higher doses actually increasing the level of viral production in monocytes (Bourinbaiar, A. S., and Lee-Huang, S., 1995, Immunology Letters 44:13-17). However, none of these reports disclose the potential efficacy of xcex2-hCG peptides in HIV inhibition in vitro or of hCG or any portion or derivative thereof in HIV treatment or prevention in vivo.
Furthermore, doses of hCG below those necessary to induce a humoral immune response have been proposed for treatment of HIV infection based on observations of therapeutic effects of such doses on cats and cows infected with feline leukemia and bovine leukemia viruses respectively (U.S. Pat. No. 4,880,626). This patent suggested use of the hCG dimer at very low doses (approximately 2 l.U. per treatment).
Lunardi-Iskandar et al. (1995, Nature 375:64-68 and PCT Application WO96/04008) reported that hCG, xcex2-hCG, as well as a xcex2-hCG carboxy-terminal peptides of amino acids 109-145 (SEQ ID NO:25) and 109-119 (SEQ ID NO:7) are efficacious in the treatment of Kaposi""s Sarcoma. However, neither reference discloses or even suggests that hCG, xcex2-hCG or xcex2-hCG peptides of amino acids 109-145 or 109-119 (SEQ ID NOS:7 and 25, respectively) have any viral anti-activity or that other xcex2-hCG peptidees have any therapeutic activity.
Finally, Harris (1995, The Lancet 346:118-119) reported that treatment with hCG improved T cell counts and physical symptoms in certain HIV infected subjects.
The present invention fulfills a needs for safe and effective therapies for HIV, wasting syndrome associated with AIDS, other infectious diseases and cancer, as well as methods for promoting hematopoiesis.
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present inventors have found that hCG preparations, xcex2-hCG preparations, certain peptides of xcex2-hCG, certain combinations of xcex2-hCG peptides linked via their N-termini and C-termini by peptide bond(s), and certain gel filtration chromatography fractions of commercial hCG preparations and human early (i.e. first trimester) pregnancy urine exhibit anti-viral activities, including anti-HIV activities. In particular, hCG and xcex2-hCG preparations and certain gel filtration chromatography fractions of commercial hCG preparations and of human early pregnancy urine, as described by way of example hereinbelow, and specific peptides thereof inhibit HIV-1 replication in vitro, inhibit HIV-1 gene-expression in HIV-1 transgenic mice, reduce plasma virus levels in SIV infected monkeys and in AIDS patients, and increase CD4+ T cells in HIV transgenic mice, SIV infected monkeys and AIDS patients. The present inventors have further found that the subjects tolerated treatment with hCG and xcex2-hCG preparations very well and that the virus did not become resistant to treatment after exposure to hCG or xcex2-hCG. The present invention fills a tremendous need for a non-toxic, long-term treatment of HIV infection and its sequelae, ARC and AIDS.
The present invention relates to proteins having a sequence of one or more portions of the xcex2-chain of hCG (xcex2-hCG), particularly proteins having the sequence of amino acid numbers 41-54; 45-54, 47-53, 45-57and 109-119 (SEQ ID NOS:3-7, respectively). The present invention also relates to proteins comprising or, alternatively, consisting of, the sequence of two or more portions of xcex2-hCG, e.g., wherein said portions are linked via their N-termini and C-termini by peptide bond(s), particularly proteins having the sequence of amino acid numbers 45-57 (SEQ ID NO:6) linked via a peptide bond at the C-terminus to the N-terminus of a peptide of amino acid numbers 109-119 (SEQ ID NO:7) or linked at the N-terminus to the C-terminus of a peptide of amino acid numbers 110-119 (SEQ ID NO:27); or a peptide of amino acid numbers 47-57 (SEQ ID NO:28) linked by a peptide bond at the C-terminus to the N-terminus of a peptide of amino acid numbers 108-119 (SEQ ID NO:29) of xcex2-hCG as depicted in FIG. 8 (a portion of SEQ ID NO:2), i.e. the peptides denoted 45-57::109-119, 110-119::45-57, or 47-57::108-119 (SEQ ID NOS:30-32, respectively). The present invention also relates to certain fractions (i.e. components of a source of hCG or xcex2-hCG isolated away from other components in the source of hCG or xcex2-hCG by a separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV and/or anti-Kaposi""s Sarcoma activity.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders associated with HIV infection based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG (preferably a source of native hCG or xcex2-hCG, i.e. a source of naturally occurring hCG or xcex2-hCG, not recombinantly produced hCG or xcex2-hCG) and therapeutically and prophylactically effective proteins containing a sequence of one or more portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The invention provides for treatment and prevention of HIV infection by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG, therapeutically and prophylactically effective peptides having a sequence of a one or more portions of xcex2-hCG, modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, and derivatives and analogs of the foregoing. The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of HIV. The invention also provides pharmaceutical compositions and methods of administration of therapeutics of the invention for treatment or prevention of HIV infection.
The present invention further relates to therapeutic methods and compositions having anti-wasting activity for treatment and prevention of wasting syndromes based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of a portion or portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The invention provides for treatment and prevention of wasting syndromes by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of hCG or xcex2-hCG (preferably a source of native hCG or native xcex2-hCG, i.e. a source of naturally occurring hCG or xcex2-hCG and not recombinantly produced hCG or xcex2-hCG), therapeutically and prophylactically effective peptides having a sequence of a portion or portions of xcex2-hCG (i.e. a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g. having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide), modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of a portion or portions of xcex2-hCG, and derivatives and analogs of the foregoing.
The present invention also relates to the use of certain fractions (i.e. components of a source of hCG or xcex2-hCG isolated away from other components in the source of hCG or xcex2-hCG by a separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV, anti-cancer activity (such as anti-Keposi""s Sarcoma activity), anti-wasting activity and/or pro-hematopoeitic activity.
The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of HIV infection, cancer, and/or wasting syndromes.
The invention also provides pharmaceutical compositions and methods of administration of Therapeutics of the invention for treatment.
The present invention also relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders in which an increase in one or more types of hematopoietic cells is desirable. The therapeutic compounds of the invention are hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of one or more portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contigous or contiguous sequences, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The present invention also relates to use of certain fractions (i.e. components of a source of hCG or xcex2-hCG (preferably native hCG or xcex2-hCG, i.e. not recombinantly produced) isolated away from other components in the source of hCG or xcex2-hCG by a separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV, anti-cancer activity (such as anti-Kaposi""s Sarcoma activity) anti-wasting activity and/or prohematopoietic activity.
The invention provides for treatment and prevention of diseases and disorders (e.g., involving hematopoietic cell deficiencies) by administration either of a therapeutic compound of the invention or of hematopoietic cells, the numbers of which have been increased in vitro by contact with a therapeutic compound of the invention. The invention thus also provides in vitro methods of expanding hematopoietic cells. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, and derivatives and analogs of the foregoing.
In a specific embodiment, gene therapy methods are provided using hCG and xcex2-hCG preparations, therapeutically.and prophylactically effective fractions of a source of hCG or xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of one or more portions of xcex2-hCG, and related derivatives and analogs to induce proliferation of hematopoietic progenitor or stem cells into which cells a nucleic acid of interest is introduced either before or after proliferation. The proliferation induced by the methods of the invention can be with or without concomitant hematopoietic cell differentiation, and, in a preferred embodiment, is proliferation followed by differentiation of the cells.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of cancers based on hCG and xcex2-hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG and therapeutically and prophylactically effective proteins containing a sequence of one or more portions (i.e., a fusion protein comprising more than one xcex2-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g., having an amino acid sequence of one xcex2-hCG peptide linked via a peptide bond to another xcex2-hCG peptide) of xcex2-hCG, and related derivatives and analogs. The invention provides for treatment and prevention of cancers by administration of a therapeutic compound of the invention. The therapeutic compounds of the invention include: hCG, xcex2-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native xcex2-hCG, therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, modified derivatives of hCG, xcex2-hCG and xcex2-hCG peptides, and nucleic acids encoding xcex2-hCG and therapeutically and prophylactically effective peptides having a sequence of one or more portions of xcex2-hCG, and derivatives and analogs of the foregoing.
The present invention also relates to certain fractions (i.e. components of a source of hCG or xcex2-hCG isolated away from other components in the source of hCG or xcex2-hCG by any separation technique known in the art) of any source of hCG or xcex2-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV and or anti-Kaposi""s Sarcoma activity. The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of cancers.
The invention also provides pharmaceutical compositions and methods of administration of Therapeutics of the invention for treatment.
The invention also provides methods of administration and pharmaceutical compositions containing a Therapeutic of the invention.
As used herein, the following terms shall have the meaning indicated.
AIDS=Acquired Immune Deficiency Syndrome
ARC=AIDS-Related Complex
BFU-E=burst forming unit-erythroid. A hematopoietic progenitor cell which is capable of producing a colony of erythroid progeny cells in semi-solid medium.
CFU=colony forming unit. A cell which is capable of producing a colony of progeny cells in semi-solid medium.
CFU-GEMM=colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte. A multipotential hematopoietic progenitor cell which is capable of producing a colony composed of granulocyte, erythrocyte, monocyte/macrophage, and megakaryocyte progeny in semi-solid medium.
CFU-GM=colony forming unit-granulocyte, macrophage. A hematopoietic progenitor cell which is capable of producing a colony composed of granulocyte and macrophage progeny in semi-solid medium.
CFU-MK=colony forming unit-megakaryocyte. A hematopoietic progenitor cell which is capable of producing a colony composed of megakaryocyte progeny in semi-solid medium.
CFU-S=colony forming unit-spleen. A multipotential stem cell with self-renewal capacity, which, upon inoculation into a lethally irradiated mouse, is capable of producing a colony (module) on the spleen. CFU-S is not a marrow-repopulating cell; it is a less primitive stem cell which does not provide long-term engraftment in an animal.
CSF=colony stimulating factor
Epo=erythropoietin
FBS=fetal bovine serum. Also known as fetal calf serum.
G-CSF=granulocyte colony stimulating factor
GM-CSF=granulocyte-macrophage colony stimulating factor
HCG=Human Chorionic Gonadotropin
KS=Kaposi""s Sarcoma
OI=Opportunistic Infection
ITP=Idiopathic thrombocytopenic purpura (a severe platelet deficiency)
PB=peripheral blood
PBMC=Peripheral Blood Mononuclear Cell